Cyclosporin A (CsA) is a widely used immunosuppressant drug. Its immunosuppressive activity occurs through the inhibition of the protein phosphatase calcineurin via formation of a ternary complex with cyclophilin A (CypA). CsA also inhibits endothelial cell proliferation and angiogenesis. This has been thought to occur through calcineurin inhibition as well. However, CsA is also a potent inhibitor of cyclophilins, a class of prolyl isomerases. Because calcineurin inhibition requires binding, and therefore inhibition of CypA, the relative contributions of calcineurin and cyclophilin inhibition in antiangiogenesis have not been addressed. We have taken a chemical biology approach to explore this question by dissociating the two activities of CsA at the molecular level. We have identified a nonimmunosuppressive analog of CsA that does not inhibit calcineurin but maintains inhibition of endothelial cell proliferation and in vivo angiogenesis. The same analog also maintains inhibition of all cyclophilin isoforms tested. We also show that a second, structurally distinct, cyclophilin inhibitor is sufficient to block endothelial cell proliferation. These results suggest that the inhibition of cyclophilins may play a larger role in the antiangiogenic activity of CsA than previously believed, and that cyclophilins may be potential antiangiogenic drug targets.

The cyclic pentapeptide cyclo(-Arg-Gly-Asp-d-Phe-Lys-) is a highly potent and selective inhibitor for the αvβ3 integrin. A related compound, cyclo(-Arg-Gly-Asp-d-Phe-Val-), is a promising anticancer drug candidate; it inhibits angiogenesis and induces apoptosis in vascular cells. We have developed an improved solid-phase synthesis based on Kessler's procedure to afford cyclo(-RGDfK-) peptide in high purity and high yield in a multi-gram scale. This improved synthesis is environmentally friendly and may be generally applicable to other related cyclic peptide syntheses.

Angiogenesis inhibitors are a novel class of promising therapeutic agents for treating cancer and other human diseases. Fumagillin and ovalicin compose a class of structurally related natural products that potently inhibit angiogenesis by blocking endothelial cell proliferation. A synthetic analog of fumagillin, TNP-470, is currently undergoing clinical trials for treatment of a variety of cancers. A common target for fumagillin and ovalicin recently was identified as the type 2 methionine aminopeptidase (MetAP2). These natural products bind MetAP2 covalently, inhibiting its enzymatic activity. The specificity of this binding is underscored by the lack of inhibition of the closely related type 1 enzyme, MetAP1. The molecular basis of the high affinity and specificity of these inhibitors for MetAP2 has remained undiscovered. To determine the structural elements of these inhibitors and MetAP2 that are involved in this interaction, we synthesized fumagillin analogs in which each of the potentially reactive epoxide groups was removed either individually or in combination. We found that the ring epoxide in fumagillin is involved in the covalent modification of MetAP2, whereas the side chain epoxide group is dispensable. By using a fumagillin analog tagged with fluorescein, His-231 in MetAP2 was identified as the residue that is covalently modified by fumagillin. Site-directed mutagenesis of His-231 demonstrated its importance for the catalytic activity of MetAP2 and confirmed that the same residue is covalently modified by fumagillin. These results, in agreement with a recent structural study, suggest that fumagillin and ovalicin inhibit MetAP2 by irreversible blockage of the active site.

TNP-470 (1), a synthetic derivative of the natural product fumagillin (2), potently inhibits angiogenesis in vivo and the growth of endothelial cell cultures in vitro. The structurally related natural product ovalicin (3) also inhibits angiogenesis but possesses potent immunosuppressive activity. The recent finding that all three drugs bind and inhibit the same target, methionine aminopeptidase 2 (MetAP2), raised the question of whether TNP-470 is also immunosuppressive and whether inhibition of MetAP2 underlies both activities of ovalicin. To address these questions, we synthesized a series of analogues of TNP-470 and ovalicin and tested them for their abilities to inhibit the proliferation of either endothelial cell or mixed lymphocyte cultures. TNP-470 and its analogues were found to possess both immunosuppressive and anti-angiogenic activities. A strong correlation was observed between the ability of compounds to inhibit bovine and human endothelial cell growth and their ability to inhibit the mouse mixed lymphocyte reaction (MLR), implying that the two activities share a common molecular basis, i.e., inhibition of MetAP2. Interestingly, ovalicin and several other compounds behaved differently in the human MLR than in either the mouse MLR or human endothelial cell proliferation assays, pointing to possible species-specific and cell type-specific differences in the metabolism or uptake of these compounds.

BACKGROUND:Angiogenesis, the formation of new blood vessels, is essential for tumor growth. The inhibition of angiogenesis is therefore emerging as a promising therapy for cancer. Two natural products, fumagillin and ovalicin, were discovered to be potent inhibitors of angiogenesis due to their inhibition of endothelial cell proliferation. An analog of fumagillin, AGM-1470, is currently undergoing clinical trials for the treatment of a variety of cancers. The underlying molecular mechanism of the inhibition of angiogenesis by these natural drugs has remained unknown. RESULTS:Both AGM-1470 and ovalicin bind to a common bifunctional protein, identified by mass spectrometry as the type 2 methionine aminopeptidase (MetAP2). This protein also acts as an inhibitor of eukaryotic initiation factor 2alpha (elF-2alpha) phosphorylation. Both drugs potently inhibit the methionine aminopeptidase activity of MetAP2 without affecting its ability to block elF-2alpha phosphorylation. There are two types of methionine aminopeptidase found in eukaryotes, but only the type 2 enzyme is inhibited by the drugs. A series of analogs of fumagillin and ovalicin were synthesized and their potency for inhibition of endothelial cell proliferation and inhibition of methionine aminopeptidase activity was determined. A significant correlation was found between the two activities. CONCLUSIONS:The protein MetAP2 is a common molecular target for both AGM-1470 and ovalicin. This finding suggests that MetAP2 may play a critical role in the proliferation of endothelial cells and may serve as a promising target for the development of new anti-angiogenic drugs.